Amphetamine (AMPH) abuse constitutes a significant public health issue with important economic and social ramifications. Treatments are hampered by a lack of knowledge of how AMPH works at the molecular level. The long-term objective of this project is to obtain an understanding of the acute pharmacological modulation of the human dopamine transporter (hDAT) activity by AMPH. Dopamine transporters (DATs) that largely control dopamine (DA) clearance are targets for psychostimulants such as cocaine and AMPH. By acting on the DAT, AMPH attenuates the DAT clearance efficiency. As a consequence, AMPH increases synaptic DA levels and enhances dopaminergic transmission with profound effects on behavior. There has been very little study of the effects of AMPH on DATs at the molecular level. This project will combine molecular biology, biochemistry, confocal microscopy and biophysics to elucidate the mechanisms involved in the acute pharmacological regulation of hDAT function by AMPH. The key issues to resolve include how AMPH causes massive release of DA via bDAT, and how AMPH causes hDAT cell surface redistribution, a novel mechanism recently uncovered by us. Both these processes affect the hDAT-mediated DA reuptake, ultimately increasing the extracellular DA concentration. The experimental plan links mechanistic aspects of the transporter function to the pharmacological properties of AMPH, namely AMPH-induced hDAT currents to AMPH-stimulated DA efflux and AMPH-induced hDAT cell surface redistribution. The proposed studies address the following specific aims: 1) to characterizethe ion, voltage and substrate regulation of the AMPH-induced hDAT currents; 2) to determine the relationship between AMPH-induced hDAT current and AMPH-stimulated DA efflux; and 3) to identify the cellular components involved AMPH-induced trafficking of the hDAT. This proposal seeks to identify molecular mechanisms of the AMPH action and to reveal new cellular targets for substance abuse therapies.